CA2118320A1 - Production of reproductive hormones - Google Patents
Production of reproductive hormonesInfo
- Publication number
- CA2118320A1 CA2118320A1 CA002118320A CA2118320A CA2118320A1 CA 2118320 A1 CA2118320 A1 CA 2118320A1 CA 002118320 A CA002118320 A CA 002118320A CA 2118320 A CA2118320 A CA 2118320A CA 2118320 A1 CA2118320 A1 CA 2118320A1
- Authority
- CA
- Canada
- Prior art keywords
- cells
- subunit
- fsh
- glycoprotein
- human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 12
- 229940088597 hormone Drugs 0.000 title abstract description 28
- 239000005556 hormone Substances 0.000 title abstract description 28
- 230000001850 reproductive effect Effects 0.000 title abstract description 10
- 210000004027 cell Anatomy 0.000 claims abstract description 82
- 238000000034 method Methods 0.000 claims abstract description 19
- 230000001105 regulatory effect Effects 0.000 claims abstract description 8
- 210000004739 secretory vesicle Anatomy 0.000 claims abstract description 6
- 230000001817 pituitary effect Effects 0.000 claims description 13
- 108090000288 Glycoproteins Proteins 0.000 claims description 10
- 102000003886 Glycoproteins Human genes 0.000 claims description 10
- 108020004414 DNA Proteins 0.000 claims description 8
- 230000003248 secreting effect Effects 0.000 claims description 5
- 238000004113 cell culture Methods 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 2
- 210000004102 animal cell Anatomy 0.000 claims 2
- 239000001963 growth medium Substances 0.000 claims 1
- 241001465754 Metazoa Species 0.000 abstract description 4
- 102000009151 Luteinizing Hormone Human genes 0.000 description 23
- 108010073521 Luteinizing Hormone Proteins 0.000 description 23
- 229940040129 luteinizing hormone Drugs 0.000 description 23
- 108090000623 proteins and genes Proteins 0.000 description 19
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 15
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 15
- 229940015047 chorionic gonadotropin Drugs 0.000 description 13
- 235000018102 proteins Nutrition 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 230000013595 glycosylation Effects 0.000 description 10
- 238000006206 glycosylation reaction Methods 0.000 description 10
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- 239000013598 vector Substances 0.000 description 10
- 150000001413 amino acids Chemical class 0.000 description 7
- 239000000539 dimer Substances 0.000 description 7
- 239000000833 heterodimer Substances 0.000 description 7
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 6
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 108700024394 Exon Proteins 0.000 description 5
- 230000004075 alteration Effects 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- OHCQJHSOBUTRHG-KGGHGJDLSA-N FORSKOLIN Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 229920001542 oligosaccharide Polymers 0.000 description 4
- 150000002482 oligosaccharides Chemical class 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 102000006771 Gonadotropins Human genes 0.000 description 3
- 108010086677 Gonadotropins Proteins 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 239000002622 gonadotropin Substances 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 101100230376 Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) celI gene Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 description 2
- 102000003864 Human Follicle Stimulating Hormone Human genes 0.000 description 2
- 108010082302 Human Follicle Stimulating Hormone Proteins 0.000 description 2
- 206010062767 Hypophysitis Diseases 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- OHCQJHSOBUTRHG-UHFFFAOYSA-N colforsin Natural products OC12C(=O)CC(C)(C=C)OC1(C)C(OC(=O)C)C(O)C1C2(C)C(O)CCC1(C)C OHCQJHSOBUTRHG-UHFFFAOYSA-N 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 230000000580 secretagogue effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- CRBHXDCYXIISFC-UHFFFAOYSA-N 2-(Trimethylammonio)ethanolate Chemical compound C[N+](C)(C)CC[O-] CRBHXDCYXIISFC-UHFFFAOYSA-N 0.000 description 1
- IVQOFBKHQCTVQV-UHFFFAOYSA-N 2-hydroxy-2,2-diphenylacetic acid 2-(diethylamino)ethyl ester Chemical compound C=1C=CC=CC=1C(O)(C(=O)OCCN(CC)CC)C1=CC=CC=C1 IVQOFBKHQCTVQV-UHFFFAOYSA-N 0.000 description 1
- 241001514645 Agonis Species 0.000 description 1
- 241001133287 Artocarpus hirsutus Species 0.000 description 1
- 102000005427 Asialoglycoprotein Receptor Human genes 0.000 description 1
- 241000713842 Avian sarcoma virus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101800005309 Carboxy-terminal peptide Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241000501458 Cultus Species 0.000 description 1
- 206010011732 Cyst Diseases 0.000 description 1
- 101100536354 Drosophila melanogaster tant gene Proteins 0.000 description 1
- UOACKFBJUYNSLK-XRKIENNPSA-N Estradiol Cypionate Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H](C4=CC=C(O)C=C4CC3)CC[C@@]21C)C(=O)CCC1CCCC1 UOACKFBJUYNSLK-XRKIENNPSA-N 0.000 description 1
- 241000735445 Hetrodes Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 241001237728 Precis Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102000002067 Protein Subunits Human genes 0.000 description 1
- 108010001267 Protein Subunits Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 101100227766 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FSH3 gene Proteins 0.000 description 1
- 102400001107 Secretory component Human genes 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 102000030621 adenylate cyclase Human genes 0.000 description 1
- 108060000200 adenylate cyclase Proteins 0.000 description 1
- 108010006523 asialoglycoprotein receptor Proteins 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 150000001508 asparagines Chemical class 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000002844 continuous effect Effects 0.000 description 1
- 208000031513 cyst Diseases 0.000 description 1
- 230000022811 deglycosylation Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 229940094892 gonadotropins Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- -1 hereto Proteins 0.000 description 1
- 230000002621 immunoprecipitating effect Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- LQRJAEQXMSMEDP-XCHBZYMASA-N peptide a Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](C)C(=O)NCCCC[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)C(\NC(=O)[C@@H](CCCCN)NC(=O)CNC(C)=O)=C/C=1C=CC=CC=1)C(N)=O)C(=O)C(\NC(=O)[C@@H](CCCCN)NC(=O)CNC(C)=O)=C\C1=CC=CC=C1 LQRJAEQXMSMEDP-XCHBZYMASA-N 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- NQLVQOSNDJXLKG-UHFFFAOYSA-N prosulfocarb Chemical compound CCCN(CCC)C(=O)SCC1=CC=CC=C1 NQLVQOSNDJXLKG-UHFFFAOYSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009450 sialylation Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000019635 sulfation Effects 0.000 description 1
- 238000005670 sulfation reaction Methods 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Endocrinology (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Reproductive Health (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Materials For Medical Uses (AREA)
- Saccharide Compounds (AREA)
Abstract
Improved methods for recombinant production of human reproductive hormones are disclosed. These methods involve the use of animal-derived cells that contain regulated secretory granules as host cells for expression systems capable of expressing DNA encoding human reproductive hormones or their .beta. subunits.
Description
- WO93/21~47 2 1 1 8 3 2 0 PCT/US93/~051 IMPROVED PRODUCTION OF REPRODUCTTVE HORMONES
Thi invention was made with gover~ment ~upport under NIH Contract No. N01-HD-9-2922 awarded by ~he National In~titutes of Health. The U.S. ~overnment has S cer~ain ri~ht~ in:this invention.
~: : Te~-Jhci~ i91~
The invention relates to methods for rec~mbinant production of human reproductive hormone~
with glycosylation patterns more clo~ely reIated to native patterns than generally obtainable in transformed cells.~ In part~icular, it concerns production of :recom~i~ant hormone ~nder conditions which result in e:fficient production and; ecretion and which regulate the gly~osyla~ion~::pattern of the protein.
Human reproductive;function is controlled in part:~y~:f~amily of heterodimeric human:glycoprotein.
ho~mone~whi~h:~haYe a~co ~ o~ a:~æubunit, but differ in their~hormone-~pecific ~ subunit~.:: The family i:ncludes 20~ ollicle~-st~imulating:~hormone (FSH3, luteinizing hormone (LH)~ thyrotr~pin~o~thyroid-stimulati~g ho~mone (TSH), and~human chorionic gonadotropin (CG)~. ~In all cases, the subunit i~ a 92 amino acid glycoprotein with two canonical glycosylation sites,at,the asparagines located :25 ~::at~po~ition~ 2~and~78. The ~ subunit~ are also glycopr~teins~: in addition:to the~N-linked glyco~ylation exhibited by:the:~;chains:of all four~:hormones, human CG
contain~ four~mucin-~like O-linked~oligosaccharide`s attached to a car~oxy-te~minal extension unique ~o this ;: 30 ~ hormone~ The rele~ance of the O-linked;glycosylation is :: not, apparently, related to the secretio~ and as~embly of ~: i : :
W093/~19~7 ~ 1 1 8 ~ 2 0 P~T~S93/o40~1 ``
Thi invention was made with gover~ment ~upport under NIH Contract No. N01-HD-9-2922 awarded by ~he National In~titutes of Health. The U.S. ~overnment has S cer~ain ri~ht~ in:this invention.
~: : Te~-Jhci~ i91~
The invention relates to methods for rec~mbinant production of human reproductive hormone~
with glycosylation patterns more clo~ely reIated to native patterns than generally obtainable in transformed cells.~ In part~icular, it concerns production of :recom~i~ant hormone ~nder conditions which result in e:fficient production and; ecretion and which regulate the gly~osyla~ion~::pattern of the protein.
Human reproductive;function is controlled in part:~y~:f~amily of heterodimeric human:glycoprotein.
ho~mone~whi~h:~haYe a~co ~ o~ a:~æubunit, but differ in their~hormone-~pecific ~ subunit~.:: The family i:ncludes 20~ ollicle~-st~imulating:~hormone (FSH3, luteinizing hormone (LH)~ thyrotr~pin~o~thyroid-stimulati~g ho~mone (TSH), and~human chorionic gonadotropin (CG)~. ~In all cases, the subunit i~ a 92 amino acid glycoprotein with two canonical glycosylation sites,at,the asparagines located :25 ~::at~po~ition~ 2~and~78. The ~ subunit~ are also glycopr~teins~: in addition:to the~N-linked glyco~ylation exhibited by:the:~;chains:of all four~:hormones, human CG
contain~ four~mucin-~like O-linked~oligosaccharide`s attached to a car~oxy-te~minal extension unique ~o this ;: 30 ~ hormone~ The rele~ance of the O-linked;glycosylation is :: not, apparently, related to the secretio~ and as~embly of ~: i : :
W093/~19~7 ~ 1 1 8 ~ 2 0 P~T~S93/o40~1 ``
the ho~mone ~M~tzuk, M.M. et al. Proc Natl Acad Sci USA
(1987~ ~4:6354-~35~).
Genomic and cDNA clones have been prepared : corresponding to the human ~ chain tBoothby, M. et al. J
~5~ ~m (1981~ 256:5121-5127; Fidde~, J.C. e~ al. Mol A~p Genet (1981) 1:3-18). The cDNA and/or genomic ~; ~equences of the ~ subunit~ have also been prepared. For CG, the ~-encoding DN~ i8 described by Fiddeg, J.C. et al:. ature (1980) 286:684-687 and by Policastro, P. et al. J Biol Chem (1983) 258:11492--11499. For luteinizing hormone, hereto, uch description is by Boorstein, W.R.
et al. Nature (1982~ 30Q:419-422; and-for TSH by Ha~a~hizaki, Y. ~t al. FEBS ~ett ~1985) 188:394-400 and y Whitfield, G.K. et al. in "Frontiers in Thyroidology", 15~ 9a6)~ Medeiro~-Nato, G. et al. (eds) page~ 173-176, Ple~um Press, NY. These DNA segment~ ha~e been expre~sed rec~m~inantly, and biologically active material has been :pr~duc~d.: : ~ :
The genomic sequence~encoding FSH~ chain was 20:~ u~ed to construct a~:~recombinant:e~pre~sion ~ec~or ~A~
c~:aini~g the complete ~:chain coding 3equence as escribed in~P~T application W0 ~6/04589, publi hed 14 Augu~t l9~. I~ addition genomic clones for human FSH-~have~been prepared~by others (Wàtkins, P.C. et al. D~
2s:~ 987)~6:2os~-2l2;:Jameso~ J.L- et a~ J y~LL~L
(19883 c806-8~15; Jame80n, 3.~. et al. J Clin End~orinol :M~ab (19~6):64::319-327;~Glaser, T. et alO Nature (1986) 882-887):. PCT:~applira~ion~WO 90/09800 describes the - expresRion of human~FSH in Chinese ham$ter o~ary cells.
30~ ~The bovine ~-FSH gene ha~also been~o~tained as disclosed in Maurer,:R.A. et al. DNA (1986) 5:363 369; Kim, K.E. et a~. DNA (1988j 7:227-333. ~ :
~: The above-refarenc d PCT application W0 ~ ~ 90/09800 discloqes a number of e~pr~ssion systems for : :: :
WO93/21947 2 1 1 ~ 3 2 0 PC~ 93/04~S1 human reproductive hormones including their a subunits and ~ subunits~ In addition, this application describes ~: certain m~teins of the ~ and ~ subunit that are useful by virtue of their alteration of ~ecretion characteristics or ~lycosylation patterns~ However, the expression systems de~cribed specifically in the above-referenced PCT applioation are limited to murine cell9 and Chine~e hamster ovary cells. The pxesent application describes the uEe of expre sion systems of the type disclo~ed in the abo~e-referenced application in celIs containing e::retory granules, e~pecially pituitary-derived cells.
: ~ : Thi : results in mature forms of the ~ subunits or :heretodlmers glycosylated in a manner analogous to their native ~orms, as:~well as an enhanced capability of the :15~ cell~:to~ecrete;the hormone.
Di closure of~the InventiQn :The invent:ion prQ~ides:cultures which are capable of ~e~reting forms of human gonadotropins, luding~their~:indiYidual ~ subunit , and including .~-~
~muteins of~ehe~hormone and subunit~ which havegl ~ osylation patter~s;similar to the~natively produced materi~ls and~which~are ~apable of~beln~ ~ecxeted into :t~e~medium.~ Secretion ints the:medium~greatly ea es the p~oce~ :o~ purification of the;~ormo~e:~pro:duced and the glyco ylation~mimicking that of the nati~e ~ubstance permi~s ~e~ter predictability of behavior i~ vivo in ~iew ; of the accumulation of data with regard to the nati~e materials.
; :: : Thus, in one:aspect, the invention is directed-to a me:~hsd to produce human~reproductive hormones na~lotropins~ or their ~ subu~its~xecombinantly, which method comprises culturing cells derived from animal tissue which:cells contain secretory granules, that ha~e :: : :
~: :
:~
W~93/21947 2118 3 ~ O PC~/USg3/~051 been transformed with an expression ~ector capable of expressing a DNA encoding said human reproductive hormone or ~ ~ubunit thereof under conditions where aid expression is effected and xeco~ering the hoxmone or : 5 subunit ~rom the ~upernatant of the culture. In another a~pect, the invent~ion is directed to the cul~ures of `these transformed cells useful in the method of the inventio~. :
Brief Desc~1E~isa~ _f the Drawinqs ~: 10 ~igure 1 ~hows a diagram of the con truction of : the human ~ subuni~ minigene, and vectors for it~
:: ` ;
xpre~io~
Figure 2 ~hows a diagram of the construction of the extended form of FSH ~ subunit.
15~ igure 3 ~hows a photocopy of a gel run on 35~5-cystei~e labele~ hH-~ subunit and 35S04 labeled or ;S-cys~eine:labeled LH dimer recombinant super~atan s.
Flgure 4, panel A is a photocopy of a gel 1 on lys~ate~ and mRdia of:~su1fate labeled~hH-e ~ ressing cel~
O~ in~the~pre e~ce and~absence of ~ecxetagogue; panel B
ohow the results of treatment with Endo~P.
Modes~of:~:carrvl~3L~~ Y5~S~.~
: As~used~erein, human ~ subunit, and human FSH, ~H, TSH, an~ ~G-~ subunits as well aæ the heterodimeric ~' 25 forms ha~e in ge~eral their conventional defi~itions and referred to ~he proteins~having:the amino acid equences known in the~art per~se,: or allelic variant~ thereof, deliberately constructed muteîns the}eof maintaining the activi~y of the nati~e:protein:~regardless of the g~lyco~ylation~pa~te~n exhibited, or:~mut~ant ~orms thereof having at lea~t 90~` homology with the native forms.
'Huma~ reproductive hormonea:" or gonadotropinsll and :
~ ~ .
W093~ 47 2 1 1 8 3 2 0 PCT/~S93/W~51 S-subunits thereo refer~ to the~e four heterodimers (or their muteins) and subunits thereof.
: "Native" forms of these hormone~ or ~ubunits are those which have the amino acid sequences i~olated from human ti~sue, and have these known sequences per ~e, or their allelic ~ariants.
"Mutein" forms of these proteins are tho~e which have deliberate alterations in amino acid sequence produced h~, for example, site-~pecific mutagenesis or by other recombinant manipulations, or which are pr~pared ~ynthetically. The e alterations re~ult in amino acid sequences wherei~ the biological activity of the subunit is retai~ed a~d/or wherein the subunit has at least 90 homolo~y with the nati~e fo~m.
~ For~example~ a preferred mutein of the subuni~for use in:a~tagoni ts of the varlouR
heterodimer :~has~ alterations in the amino acid~ of po~itions:88-92:.: ~ ;
A part~i~ularly preferred~mutein of FSH-~ or :20 ~ or ex~m~le,~is:~an "extended" FS~-~ or~H-~ whe~in th~ amin~acid~se~ue~ce compris~in~the carboxy-terminal peptide~(CTP)~:of hCG iB fused to~the~car~oxy terminu of PS~-~ or~ :. A~us~d~herein, "CTP"~refers to the "extra" equence ;at~the~C-termi~us::of:the CG-~ peptide a~
2s~ compar~d~to the other~related hormones.~ The length of the effecti~e CTP:a ~:~ompared to the other ~ subunits may vary slightly'but it extends from roughly amino a~id ; 112-118 of CG~t~residue~145 at;the~C-terminus. The preci e length::of:~TP~in the:constructs here`in will be 30 ~clear from the content. : ~ :
In~the~usi~ns~describe:d~herein, native CTP can : be u3ed or a~"varian~":thereof.~ By i'variant" is meant a con~ervative analog of the peptide~residu~s from ~bout 2-l18 to: 145, i.e~. thi~ sequence wherein about 1-5 :: : : ~ : :
:
., ,, . , . ., =..
W~ g~/21947 2 1 1 8 3 2 0 PCI/US93/04~5~
amino acid~ of the sequence are altered without æubstantial change in propertie~. Often this variation results ~imply from muta'cion to obtain appropriate restriction sites.
Although it i~ recognized that glycosylation pattern ha~ a prof ound inf luence on activity both qualitatively and quantitatively, for convenience the :: : terms FSH, ~I, TSH, and CG ,B subunits refers to the . amino : : acid sequence characteristic of the peptides, as does : 10 ~ubunit". When only the ~ chain--is re~erred to, the terms will be, for example, FSH-~; whèn the heterodimer is referred to, the simple term "FSH" wi11 be u~ed. It will be clear from the con'cext in what manner the glyco ylation patterIl is affected by, for example, s ~ 5~ recombinant expxession ho t or alteration in the glycosylati~ n ites. Forrns of the glycoprotein with ;: Epecified glycosylation patterns will be so ~oted .
As :used herein, the a subunit "minigl~ne" refers to ~ithe géne constru~tion disclosed ~ in PCT applicatiorl WO
o ~ 90/0980:0 in the description of the coxl~;truction of ,.~, 12/CGa or p~l2/cY. ~This "minigene" i5 characterized by rete~tion only ~of 'one iiltron sequence such as that between exon III and~ exon IV, all other introns ha~ring been~;deleted:.~In the particular construction described, t~e~N-terminal:co~ing sequenc~s which are derived from ~: exon II and a~portion of exon III are ~pplied from cDNA
: a~d arè ligated directly through an ~baI re triction site : into the coding se~uence ~f exon III 80 that the i~tron~
between exons I and II and between exons II and III are absent. Howe~er, the intron between:exons III and IV as well as the signals 3~ of the coding ~equence are :: retained. The resulting minigene can con~eniently be ~:: inserted as a BamHI/BglII segment. Other means for ~ construction of a comparable minigene are, of cour~e, :: :
wo 93/2lgq7 2 i 1 8 3 2 0 P~T/U~93/OqOSl possible and the definition is not restricted to the parti~ular construction wherein the coding se ~ ences are ligated through an XbaI site. However, this is a con~enient means for the construction of the gene, and S there i~ no particular advantage to other approaches, such as synthetic or partially synthetic preparation of the ge~e. The definition includes those coding se~uences for the ~ subunit which retain one intron such as t~at between exons III and IV but no other introns.
~: 10 A~"tran~formed" recombinant host cell, i.e., a cell ~transformed~ with the recombinant expression ~ystem~ of:the inve~tion, refers to a host cell which has bee~ alt~red to contain this expre~ ion sy~t~m by any conveni~nt manner of introducing it, including ~:~:; 15 tra~sfection, viral infection, and 80 forth.
: "Tra~sfonmed" refer~ to cell~ co~taining this expression system wh~ther the ~ystem is integrated into the chro~osome or is~extrachromosomal. The "transform d~
cells:may either be stab~e with re~pect to inc~usion of 20:~ ~the expression system or not. In~short, recombînant ~st cells~"transformed" w~ h the expre i~n ~y~tem-of the : invention ref rs:t~ cells which include thi~ expres~ion ystem as a; re~ult~ of their manipulation to include it, when they na~i~el~:do not, regardle~s of the ma~ner of 25 ~: ef~ect~ing ~his inco~poration.
Expression ~y~tem" re~er to a DNA sequence which i~cludes a coding sequence to be expre~ed and : tho~e accompanying control D~A sequences nece~ary ko effect the expre~sion of the coding ~e~ue~ce. Typically, 30~ these control inc1ude a promoter, termination regulating equ~nce~, and, in ~ome cases,~an ~perator or other mechani m to regulate expression. Th co~trol sequences are tho~e which are designed to be functional in a particular target recombinant host cell and ~herefore the WO93/21947 . . PCT/US93/04nSI
host cell must be chosen ~o ~s to be compatible with the control sequences in the constructed expression system.
As used herein "cells~ 'cell cultures", and : "cell lin~" are used interchangeably without particular attention to nuances of meaning. Where the di~tinction etween them is important, it will ~e clear from the context. Where any can be meant, all are intended to be include~.
Certain cells are known to contain dense-core ~secretory gr~ules and to secrete proteins through a regulated pathway, which can be stimulated by certain ~ubstances, for example, forskolin. These cells or cell Iine , deri~èd from appropriate animal ti~ues, are the ho~t celIs of the invention. Included among cuch cells :l5~ are cells of~the secretory component~ of ~he hormo~e y tem~uch;as~:the:pituitary, ~ i let cells, and cells of the~adre~al co~tex:.:~ Particularly preferred in the method of; the invention are pituitary-derived cell~.
o~ tent~with the foregoing paragraph, "cell~
2:0~ ;deri~ed rom:pituitary" refers:the cellR or cell lines ;which~are cultured~fr~m pituitary tissue:derived from ani~al~ pe:cies,; in particular;mammalian specie~, and more paxticulàrly,:~hu~ar or:murinç;pituitaries. Illustrated herein i~the G~3 murine~cell~line described by Tasjian, 2~5~ :J.,~ (197:9):~ 5~7.: However, other lines derived:from~the:pituitary~are~al~o ~nown and obtainable rom p ~ lic depo~itories. In addition, cells deri~ed directly from~pituitaries:may~be~:~u~ed. ~ ~
Cell~s containing ~ecret:ory::granules provide an ;30~ environment:more like that :o~ ~pituit~ry cells as opposed to CHO cell~. This re~ultæ in glyco~ylation patterns : : more like the nati~e orms~:and enhances ~ecretion efficiency of proteins~to be secreted.~ ~Production of the : gonadotropin~ in such cells,~containing dense-core ::: :
~-.. W0~3/~1~47 ~ 3~ o P~T/U~93/040~1 secretory granules capab~e o secreting proteins through a regulated pathway thus results in the production of secreted forms of these materials with glycosylation patterns similar to those found in the native hormones or subunits. In particular, these forms of the hormones or subunits ~u~h a5 LH which retain labeled sulfur ~upplied in the form of sulfate do so when produced in these cells ~: indicating that ~ulfated glycosylation units are presen~
in the~e forms. ~abeled SO~ 2 i~ not incorporated into : : 10~ ~H produced in CHO cel}s or murine Cl27 cells.
~: While FSH is normally sialylated, the ~ subunit of ~H is normally sulfated. Sialylation of N-linked : : o1igosaccharide~ has long been recognized to pxotect :circulating gonadotropin from clearance by hepatic 15 ~ asialoglycoprotein receptor In contra~t, the pre~ence , ~ ~
` ~:: : ~ o~ulfate on ~H lead to a rapid;clearance of the h~ ormone; ul~ated b~vine LH h~s a 4-5 fold faster ~et~bolic clearance compared to the corre~ponding ialy1at~d~re~ombinant bovine L~I. Sulfated LH is removed 2;0 ~ om~ci~culation upon binding to a ~pecific receptor p~
;the~surface:o~liver epithelial cells which re~ognizes a specific ~ulfa~ed trisacchari~e in ~he~N-linked glycosylation.~;~ De~ulfated ~H~behaves simi~arly to ;:sul~ated~hX~in~recept~r binding:and ignal transd~ction ; 25~ in~:MA-~lO cel~ls,~but~hi~h in~ vivv do9es are requixed to ti~ulate o~ulation,~pre~umably be au e ~he circulatory half life is altered by sulfation. It is believed that clinical u~e of ~R:in human~or animal reproduction wilI
requlre the hormone~in~sulfated form.
, 3:0~ ExPression Vectors ~ ~
:~ ~To co~struct ~uitable:expression vectors for , use in the ~ecretory cells of the invention, a convPnie~t co~struct,~ iIlustrated for the ~ subunit minigene i5 ~::
:
WO93/~1~47 21 1 832 0 P~/U~93/~051 :~
reproduced from the above-referenced PCT application as Figure 1. As shown in this figure, and as more fully explained in the above-referenced app~ica~ion, the se~uence encoding the ~ sllbunit is prepared as a minigene wherein the portions of the peptide encoded by exons II-III are fused but separated from exon IV. Thi~ construct : is ligated into the host vector pM2 under the control of the long terminal repeat ~hown in Figure 1 to obtain : pM2/CG~ or to obtain pM~a which contains an additional ~0 insertion site for another coding D~A. As further shown in Figure l, the BamHI site contained in the ho~t vector pM2 can be u ed to accommodate the ~ subunits of the human r productive proteins for expre~sion of the~e subunit per~ se, a well a~ providing a separate ; 15 ~expr~ssio~ vector for concomitant production of the a and subunit by rells tran~fo ~ ed by both vectors.
Thus, the:~ sub~nits per se may be produced by ligating the coding DNA ~into pM2 a~ ~hown, or the heterodimer may be produced by cotra~sfo ~ ation of pM2 :~aontainîng ~A;encoding ~ ~ubu~it with pM2/CG~
Production of ~he;heterodimer i~ preferred. The heterodimer may:also~be~produced from the;single vector wherein the ~ in ert i liyated into the Bam~I site of pM2/a~créated~by ligation of EcoRI-digested pM2 with 25~ ~co~ dige~ted modified pM /CG~ -:~: The ~oregoing construc~ions are, of cour e, ' ~ merely illu~trative of expression vectors or ~ystems~
: : which an be constructed for the prod~ctio~ of ~ subunits or the correspondi~g:heterodimeric hormones. Alternate ;: : 30 :control ~equences incIuding, for exa~ple, different : ~ promoters, can be ligated to the coding seque~ce of human ubunit~ or the a minigene to effect expression. A
~:~ : ~ariety of control sequences is known in the art, and methods to ligate the ~ subunits or ~ minigene coding ~ :
W093/21947 2118 3 ~ ~ PCTlUS93/~51 sequence (or other ~-encoding construct) are of course also available~ Suitable mammalian promoters include the early and late promoter~ from SV40, or other viral : promoters such as those derived from polyoma, adenovirus : 5 2, bo~ine papilloma virus or avian sarcoma viruses.
: ; : Suitable viral and mammalian enhancers can also be used.
~ As ~et forth in the Background section above, ;~ the recovery of the genes for the various ~uman :
reproducti~e hormones, including their ~ subunits, has : 10 already been described. The genes can be recovered from native ~ource~ as described in the art, or the genes can be entirely or partially synthesized u~ing ætandard solid phase oligon~cleotide ~yntheæis techniques as described, :;for example, ~y Nambiar, K.P. et al. Science (~984) 5;~2 1299 or by Jaye, E. et al. J Biol Chem (19843 259::6311. The ~echniques are now commercially ;ava~ilable. It~i~ evident, of cour~e, that not only th~
specific native nucleotide ~equence~ can be employed, but al~o nu:~eotide equences:employing codons which are o~ degenerate~with thes~e~,~ as~well:~a~s~allelic~forms. .~-~: AB f~rther~de~cribed~in the aboYe-referenced PCT~applicatio~n,:muteins of the:variou~ hormone~ can be o~tained~:which:have~agoni~t or antagoni~t activity.
These~mutein-encoding~genes~may~be inserted into the 25 :~ rele~ant ho t:vectors in a manner~:similar to that de~cribed for~the nati~e for~s. Of parti~ular intereæt : are mutein wherei~ the:~ subunits of LH, TSH or FSH are : extended b~ the carboxy:terminal peptide native to :: : chorionic gonadotropin.: One ~uch ~on~truct is 30~ lu~trated in Figure 2 for human~F5H. In addition, more than one of t~e ~TP~uni~s may be used to extend the : chains.
: , WO93t21947 ~ 3 ~ O PCT/U~g3/~051 In general, the selected expression v~ctor or vector~ is transfected or otherwise transformed into the host cells of the in~ention using procedures similar to ~:S those u~ed generally ~or mammalian cells. The techniques ~: ~for tran~formation are ~ubstantially those applied with re~pect to Chine~e hamster o~ary cells, and ~y~tems for marker~ for selection of succe~ful transformants are : ~- a1so ub~tantially~identical. Cell culture conditions for the ~ransfonmed cells ma~ be---modified as is generally under tood to accommodate ~he particular need~ of the selected ho~t cell line which will contain reyulated secretory g~anuleæ; however, culture conditions are ` 8ub8ta~tially similar to tho e u ed for ~HO cells a 15~ well. ~
In an illustrative approach to transfection and cultu~e, the:~subunit-encoding yene i9 inserted into pM2 an~then transfected alone or along with~pM3/CG~ into the ce~ ;appropriate for the method of the:invention which `O~ con~ain regulated ecretory granu1es or into Chinese ~-~iham~ter~o~ary:cells as~a contr~l,~a~ described by Matzuk, M.M.~ et~al., ~b=~ g~bS~ L (;1987) 8~:6354-6358;
Matzuk~ M.M.~ e~al., ~ 5~LL~al:(19883 106:lO49-1059.
Succe~ful transformant :are ~elected in 0.25 ug/ml G4l8, 25:~ and~e~pre sion~may~be~detected ~y~immunoprec:ipitation of : metabolically labele~ cells to elect:monomer- and dimer-secreting cell lineB.
- Both~stably tran~fectQd~hosts ~ontaining resulated secretory granule and:~tably tran~fected ~HO
: 30~ cell~;lines are maintained in "medium-~l" (Ham'~ FlO medium æupplemente~ with 1~5% horse serum`and 2~5~ fetal calf :
erum, penici1lln (100 U/ml), streptomycin (lO0 ~g/ml), :glutamine (2 mM) and 0.125 mg/ml G~l~ in a humidified 5 ,~
:C2 incubator.
~ .
.. WO93/21947 2 1 18~ ~ P~T~US93/0~51 Labelinq Both cells of the invention and C~0 cells are plated at 300,000-350,000 cells per well into 12-well di~hes in 1 ml of F-10 medium containing 15~ horse serum ~ ~ S and 2.5~ fetal calf serum and pr~plated 3-4 days. For : : : continuou~ labeling, cells are washed twice with ; cysteine-frèe "medium-2" (medium~1 supplemented with 5~
dialyze~ calf ~erum) and labeled for 6 hours in 1 ml of cysteine-free~medium 2 containing 20 uCijml la~eled 0 ~ cyseeine. ~For pulse cha~e experimentæ, the cells are washed twice and preincubated for 1.5 hours in cy~teine-~ree medi~m-2,~fo~1Owed by a 20 minute labeling in cy teine-fre@~medium-2 contai~ing 100 uCi/ml labeled :cy~teine,~ then washed twice with medium 2:containing 1 mM
5~ unlabeled~cyst:eine and incubated in the unlabeled medium.
a~elin~ ~periment~ using incorporation o 35S:-labeled sulfate~are~aonducted similarly.
In~more detail the medium`and cell ly~ate are prepare~, immunopre~ipitated, and:treated~as described by 2~ ~rle~6,~ c.~ et al. ~ J e1l ~ioI~ 87): 104:1173~
and~ he~Matzuk~P~a~ paper cited~above. Anti~era agalns~ G~ ~H~ :FSH-~ and T9H~ and the ~ ~u~unit are preparèd~by~tandard:methodsi:~antisera~generated against CG-~cro~s~-~reaetæ ~ully~with ~ a~d~can be u~ed to 25;~ ;detect LH-~as well.~ ~For ~haracterization of the : immun~precipi~ate ~n SDS ~els,~ SDS~polyacrylamide gel are soaked for~10 minutes in l M~sodium ~licylate, d~ied~ and;autoradiographed~with preflash film,: ~nd scanned, i~: desired, with:an LKB~Ultra~can X~ lasex:
3~0:: ~den~itometex.~
: As~a~cor.trol, the results~:of production of : human FSH;in:CHQ cells is asseæsed by l~elin~ with ~: ' :
::
WO93/21947 2 i 1 8 3 2 0 PCT/US93/~1 cy teine. The expres~ion systems described above for human FSH-~ inserted into pM2 for expression of F5H-~alone or into pM2/~ for expre~sion in tandem with the ~
subunit were transfected into CHO cells and stable clones shown to e~pregs the ~ subunit or dimer were continuously labeled with 35S-cy~teine for 6 hr. Yields of secreted FSH of approximately 1 mg/106 cells/24 hr cultured in 1 L
of medium were obtained.
: The proteins secreted into the media and from cell ly~ates were immunoprecipitated with appropriate anti~era and resolved on SDS-PAGE. The results obtained : com~are the behavior o~ similar transformants expressin~
the gene for human C~
Gel~ from 6 hr labeling ~how that in the ~abQence of ~ subunit FSH-~ i9 retained in the ly~ate, while when the:~ ~BUbUnit i~ pre~ent, the dimer is formed ;and efficie~tly ~ecreted into the medium.
When the cells are pulse~labeled wit~
35S-~cysteine~for~20~min and:chased with unla~eled 2~ ¢y~teine for up~to 12 hr,~result~ for the ~ subunit of~CG
show~the~half-life in lysat~es and appearance of-CG-~ in t~e~medium are~identical;at about 2~hr and almo&t all the secre~ed~ ubunit can~:be recovered:. A lower molecular weigh~ present ~in the:~medium a~parently due to the 25~:~ dif~ereDces; n the extent:of:glycosylation at the 2 APn-:: linked~glyco ylation:sites on CG-~ and is unigue to this ubunit. FSH-~ alone is secreted much les~ efficiently and, a~ does~G-~, di~appear~ from the cell lysates af~er a~out 5 hr; 1 ss than 20~:is recovered in the medium : 30 after 1~ hr.
Simi:larly~:to the ~ subunit~ of:LH and TS~, FSH-alsne is ine ficiently secreted and slowly degraded intracellularly. : Howe~er, the pre~ence of the ~ subunit tabilizes and enhances the secretion of the ~ subunit W093/21947 2 1 1 ~ 3 2 O PCT~US93J04~51 -l5-for FSH. The half-life for diRappearance from the lysates was about 90 min, and 90% was recovered in the medium after l2 hr. This behavior is similar to that shown for TSH, but different from both CG and LH.
I~ i~ apparent from the foregoing results that dif~iculties with ~ecretion are experienced or expression in CHO cells of the genes encoding the ~
: subunits alone; in ome instan~es, this secretion abili~y is mitigated by the presence o~ a vector encQding the ~
~ ~ubunit. However, in general, it appears that CHO cells are not efficient secretors~of the desired ~ subunits or correspondi~g human reproducti~e ho ~ one~
AnQther~difficulty which is apparent from experiments using sulfate label as a source of 35S is ;;l5~ that~for L~ subunit or thP LH heterod.imer, which is k~own to contain ulfated glycosylation in it6 native tate, in~orporation~of label does not take place in CHO
G~ h~s, :~a~ lea t~for LH, and~for:the ~ portion of th~ :heterodimer., which~also contain~:sulfated 2~0~ glyco~ylation~,~HO~;cel~lc sre unable to reproduce the _-~
b~ native::patte~n::for glyco~yla~io~.: Thus, it has been show~ that u~ing,~;~in~place;of ~ysteine, labeled sulfate a~:;a ~ource:o ::ra~ioacti~ity, label does not appear in the~di~er~or~ the ~ hunit of~ L~ produced in CHO cells~
:: 25 ~ ~
: The~:e~pressio~ vectors described aboveincluding :the~ ubunit~ for LH~ CG-~ or FSH-~ either with or without the~a subunit,:and~with the ~:subunit encoded on the same~or different ve~tor wexe tran formed 30~ ~into the:rat~ pituitary cell~ line GH3. After æteady s~tate : :laheling ~y either sulfate (for ~H~ or cys~eine-oriyinated ~for all~:forms) S35,~mature forms of LH, FS~
and hC~ which contained processed oligosaccharide~ were~
~: : :
W~93/21947 2 1 1 8 3 2 0 PCT/U~93/~4051 ~tored in the cells and their release was stimulated by for~kolin. The majority of the ~ ~ubunits of these hormones appeared to be Endo H sensitive and thus re~ide in the ER, but the Endo H resi~tant fraction, which i6 the mature form, was targeted to the regulatory ~ecretory :: pathway with an efficiency that was comparable between : the dimer and the ~ ~ubunit.
Figure 3 shows the result~ obtained from G~3 cells which have been tran~formed with pM2/~ into which ~: 10 the coding ~equence for hH-~ ~ubunit has been inserted into the alternate BamHI site. Lanes 1 and 2 are controls f~om ~H3 ~ell~ which are trans~ormed only ~y pM~/CG~; a i~ evident from the results, the ~ ~ubunit is found mo~tly in the cells themselve~ (C) rather than in : lS ~the ~up#rnate (S? ~n the other hand, lane~ 3 ànd 4 : repre~nt the re ults obtained b~ immunoprecipitating the :~ .
he~rodi~er labeled as de ri~ed above u~i~g cysteine or ul ate~and a~aying the cell medium. A~ ~een from these re~ult~ a large amount of the heterodimer i~ ~ecreted 20~ into~the medium and both the ~ and ~ ~ubunit are able take up }abel rom~5 sulfate.:
n an;additional e ~ eriment, cell~ expres~ing ; :LH were la~eled with~t35S]04 for 16::~rs~to allow accu~ulatio~ of the labeled hormone~ in ~he ~torage :~gran~les. To:~de~crea~e the background of ~onstitutive ~ecretion (i.e., the material not ~ecreted through den~e core granules of the reyulated pathway) following the ab21i~g, the cells were cha ed twice at 2 hr i~tervals :
befo~e timulation (desi~nated I and II in ~igure 4). ~t : 30 the beginning o the:third cha~e (III), 20 ~M of the : ~ secretagogue forskolin (an adenylatecyclase activator) : was added to a duplicate set of wells; a parallel ~et was ~ incubated without ecretagogue. The media (M) and the . W~93/21947 2118 3 2 0 PCT/US93/04~1 cell extracts (L) ~ollowing the third cha~e were precipitated ~y an~ serum and analyzed on SDS-PAGE.
In Figure 4, panel A, lane 1 represen~s the medium at cha~e I. This lane shows that LH~ and ~
subunit~ wexe labeled~by [35S]04. This initial pool is presumably a rapidly secreted, not stored pool. After 4 : ~ ~, hours (II~ and 6 hours (III) of cha~e, the constitutive pool is depleted i~ce no further material is prese~t in the media ~1~a~es 2 and 3 of panel A). Howe~er, there is stil1 a pool of ~H i~. the cell as ~hown by panel A, lane 5 which repre~ents tn~ lysate of cells not treated with : forskoli~. Thi~pool is r~.-lea~ed by the secretagogue :; into the ~edia as shown by 2anel A, lane 4. Lane 6 shows that the intr~cellular pool is depleted following ~orskoli~ treatment~
To demo~strate that radioactive ~ulfate wa~
corporated~i~to N~ ked oligosaccharide~, immunoprecipitated~35S]04 labeled LH was treated with endoglycosida~e~F which is known t:o remove c~mplex N-20~ inked 0ligo~accharides. ~The results of thiæ treatmç~are shown::in~Figure 4, panel B, lane~ I and 2.~ Upon digestion, with~Endo F, the~radioactivity disappeared fo~m~both~a~and~sl~hunits. ~anes 3 and 4 are controls to~how that~t~i~ wa~ not due:to protease activity during :25 ~ incubation.:~ The~e~lanes show the result of Endo F
treatment of LH ~ecreted:into ~he medium after labelIng : ~ with 35S-cy teine. The 35S-cy teine labeled LH subunits collapRe to~a~sin~le band orregponding to the deglycosylated;protein following Endo F dige~tionO
:~ ~ ~ The~e data show that~ LH iæ ~tored in the : ~ ~ GH3 cells~and it ~:release ls:s~imul~ted by 3ecretagogue uch ~torage and:release is not 9een in CH0 cells :~ whethex ~a~eled with S04 or cysteine), (2~ The N-linked :: :
W093~2~947 2 1 i 8 3 2 0 P~T/US93/04~1 .
oligosaccharides of LH are sulfated when produced in GH3 cells.
As~embly of the ~ 3ubunits of these hormones and secretion of the dimers is increased many-fold over that en in CHO cells. The oligosaccharide in the LH
dimer is al~o ~ulfated; howe~er, FSH is normally sialylated in~ ~o,' and thus does not take up label from S04~ ~ :
It has b~en demon~trated that complete 10 ~deglycosylation of human chorionic gonadotropin results in a hormone which retain~ its ability to ~ind to receptor, but :is no longer capable o~ eff~cting the ordinary biological:~re~pon e of the cell on which the receptor i borne. Simila~ effects of ~omplete.
15~ eglyco~ylat;io~are~o~tained with the additional reproductive;~hor~one. LH and FSH.~ Accordingly, alt~ration o the glycosylation::pattern in the ~ subunit may~;:ra ult in:alternative~propertie~. The glycosyl~tion of;the~:hormones an~:~subunits produced b~he method of 2~ he~i~ve~ however;~ more clo~ely:re~emble~ hat o~-the~native~iorm~
The:~honmone~and~oth~r~pharmace~tical of the~
presen~inYe~ion~are formulated ~or~administration u ing 2~ ~method ~generally u~der~tood in~the a~t~ Ty~ical : ; formiulation a~d ~odes of:admi~i tration are descrihed in Mack~Pu~lishing Co., Easton, PA,~ latest~edition.~The~e~formulation~ are typically ~or~ystemic:administrati~:, such as by 30~ :i~jection, but oral~f;ormulations~:or topical ~ormulations may al~o be~employed.
The choice of formul~a~ mode of admini~tration,:and do~age lèYel are ~dependent on the .
:
WO93/21947 2 ~ 1 8 3 2 0 PC~/US93/~4051 particular hormone or protein and can be optimized for the appropriate indication u ing generally recognized techniques.
:
~: :
:
::
~: :
,.
~ . : : . :
:: ~: :
:
(1987~ ~4:6354-~35~).
Genomic and cDNA clones have been prepared : corresponding to the human ~ chain tBoothby, M. et al. J
~5~ ~m (1981~ 256:5121-5127; Fidde~, J.C. e~ al. Mol A~p Genet (1981) 1:3-18). The cDNA and/or genomic ~; ~equences of the ~ subunit~ have also been prepared. For CG, the ~-encoding DN~ i8 described by Fiddeg, J.C. et al:. ature (1980) 286:684-687 and by Policastro, P. et al. J Biol Chem (1983) 258:11492--11499. For luteinizing hormone, hereto, uch description is by Boorstein, W.R.
et al. Nature (1982~ 30Q:419-422; and-for TSH by Ha~a~hizaki, Y. ~t al. FEBS ~ett ~1985) 188:394-400 and y Whitfield, G.K. et al. in "Frontiers in Thyroidology", 15~ 9a6)~ Medeiro~-Nato, G. et al. (eds) page~ 173-176, Ple~um Press, NY. These DNA segment~ ha~e been expre~sed rec~m~inantly, and biologically active material has been :pr~duc~d.: : ~ :
The genomic sequence~encoding FSH~ chain was 20:~ u~ed to construct a~:~recombinant:e~pre~sion ~ec~or ~A~
c~:aini~g the complete ~:chain coding 3equence as escribed in~P~T application W0 ~6/04589, publi hed 14 Augu~t l9~. I~ addition genomic clones for human FSH-~have~been prepared~by others (Wàtkins, P.C. et al. D~
2s:~ 987)~6:2os~-2l2;:Jameso~ J.L- et a~ J y~LL~L
(19883 c806-8~15; Jame80n, 3.~. et al. J Clin End~orinol :M~ab (19~6):64::319-327;~Glaser, T. et alO Nature (1986) 882-887):. PCT:~applira~ion~WO 90/09800 describes the - expresRion of human~FSH in Chinese ham$ter o~ary cells.
30~ ~The bovine ~-FSH gene ha~also been~o~tained as disclosed in Maurer,:R.A. et al. DNA (1986) 5:363 369; Kim, K.E. et a~. DNA (1988j 7:227-333. ~ :
~: The above-refarenc d PCT application W0 ~ ~ 90/09800 discloqes a number of e~pr~ssion systems for : :: :
WO93/21947 2 1 1 ~ 3 2 0 PC~ 93/04~S1 human reproductive hormones including their a subunits and ~ subunits~ In addition, this application describes ~: certain m~teins of the ~ and ~ subunit that are useful by virtue of their alteration of ~ecretion characteristics or ~lycosylation patterns~ However, the expression systems de~cribed specifically in the above-referenced PCT applioation are limited to murine cell9 and Chine~e hamster ovary cells. The pxesent application describes the uEe of expre sion systems of the type disclo~ed in the abo~e-referenced application in celIs containing e::retory granules, e~pecially pituitary-derived cells.
: ~ : Thi : results in mature forms of the ~ subunits or :heretodlmers glycosylated in a manner analogous to their native ~orms, as:~well as an enhanced capability of the :15~ cell~:to~ecrete;the hormone.
Di closure of~the InventiQn :The invent:ion prQ~ides:cultures which are capable of ~e~reting forms of human gonadotropins, luding~their~:indiYidual ~ subunit , and including .~-~
~muteins of~ehe~hormone and subunit~ which havegl ~ osylation patter~s;similar to the~natively produced materi~ls and~which~are ~apable of~beln~ ~ecxeted into :t~e~medium.~ Secretion ints the:medium~greatly ea es the p~oce~ :o~ purification of the;~ormo~e:~pro:duced and the glyco ylation~mimicking that of the nati~e ~ubstance permi~s ~e~ter predictability of behavior i~ vivo in ~iew ; of the accumulation of data with regard to the nati~e materials.
; :: : Thus, in one:aspect, the invention is directed-to a me:~hsd to produce human~reproductive hormones na~lotropins~ or their ~ subu~its~xecombinantly, which method comprises culturing cells derived from animal tissue which:cells contain secretory granules, that ha~e :: : :
~: :
:~
W~93/21947 2118 3 ~ O PC~/USg3/~051 been transformed with an expression ~ector capable of expressing a DNA encoding said human reproductive hormone or ~ ~ubunit thereof under conditions where aid expression is effected and xeco~ering the hoxmone or : 5 subunit ~rom the ~upernatant of the culture. In another a~pect, the invent~ion is directed to the cul~ures of `these transformed cells useful in the method of the inventio~. :
Brief Desc~1E~isa~ _f the Drawinqs ~: 10 ~igure 1 ~hows a diagram of the con truction of : the human ~ subuni~ minigene, and vectors for it~
:: ` ;
xpre~io~
Figure 2 ~hows a diagram of the construction of the extended form of FSH ~ subunit.
15~ igure 3 ~hows a photocopy of a gel run on 35~5-cystei~e labele~ hH-~ subunit and 35S04 labeled or ;S-cys~eine:labeled LH dimer recombinant super~atan s.
Flgure 4, panel A is a photocopy of a gel 1 on lys~ate~ and mRdia of:~su1fate labeled~hH-e ~ ressing cel~
O~ in~the~pre e~ce and~absence of ~ecxetagogue; panel B
ohow the results of treatment with Endo~P.
Modes~of:~:carrvl~3L~~ Y5~S~.~
: As~used~erein, human ~ subunit, and human FSH, ~H, TSH, an~ ~G-~ subunits as well aæ the heterodimeric ~' 25 forms ha~e in ge~eral their conventional defi~itions and referred to ~he proteins~having:the amino acid equences known in the~art per~se,: or allelic variant~ thereof, deliberately constructed muteîns the}eof maintaining the activi~y of the nati~e:protein:~regardless of the g~lyco~ylation~pa~te~n exhibited, or:~mut~ant ~orms thereof having at lea~t 90~` homology with the native forms.
'Huma~ reproductive hormonea:" or gonadotropinsll and :
~ ~ .
W093~ 47 2 1 1 8 3 2 0 PCT/~S93/W~51 S-subunits thereo refer~ to the~e four heterodimers (or their muteins) and subunits thereof.
: "Native" forms of these hormone~ or ~ubunits are those which have the amino acid sequences i~olated from human ti~sue, and have these known sequences per ~e, or their allelic ~ariants.
"Mutein" forms of these proteins are tho~e which have deliberate alterations in amino acid sequence produced h~, for example, site-~pecific mutagenesis or by other recombinant manipulations, or which are pr~pared ~ynthetically. The e alterations re~ult in amino acid sequences wherei~ the biological activity of the subunit is retai~ed a~d/or wherein the subunit has at least 90 homolo~y with the nati~e fo~m.
~ For~example~ a preferred mutein of the subuni~for use in:a~tagoni ts of the varlouR
heterodimer :~has~ alterations in the amino acid~ of po~itions:88-92:.: ~ ;
A part~i~ularly preferred~mutein of FSH-~ or :20 ~ or ex~m~le,~is:~an "extended" FS~-~ or~H-~ whe~in th~ amin~acid~se~ue~ce compris~in~the carboxy-terminal peptide~(CTP)~:of hCG iB fused to~the~car~oxy terminu of PS~-~ or~ :. A~us~d~herein, "CTP"~refers to the "extra" equence ;at~the~C-termi~us::of:the CG-~ peptide a~
2s~ compar~d~to the other~related hormones.~ The length of the effecti~e CTP:a ~:~ompared to the other ~ subunits may vary slightly'but it extends from roughly amino a~id ; 112-118 of CG~t~residue~145 at;the~C-terminus. The preci e length::of:~TP~in the:constructs here`in will be 30 ~clear from the content. : ~ :
In~the~usi~ns~describe:d~herein, native CTP can : be u3ed or a~"varian~":thereof.~ By i'variant" is meant a con~ervative analog of the peptide~residu~s from ~bout 2-l18 to: 145, i.e~. thi~ sequence wherein about 1-5 :: : : ~ : :
:
., ,, . , . ., =..
W~ g~/21947 2 1 1 8 3 2 0 PCI/US93/04~5~
amino acid~ of the sequence are altered without æubstantial change in propertie~. Often this variation results ~imply from muta'cion to obtain appropriate restriction sites.
Although it i~ recognized that glycosylation pattern ha~ a prof ound inf luence on activity both qualitatively and quantitatively, for convenience the :: : terms FSH, ~I, TSH, and CG ,B subunits refers to the . amino : : acid sequence characteristic of the peptides, as does : 10 ~ubunit". When only the ~ chain--is re~erred to, the terms will be, for example, FSH-~; whèn the heterodimer is referred to, the simple term "FSH" wi11 be u~ed. It will be clear from the con'cext in what manner the glyco ylation patterIl is affected by, for example, s ~ 5~ recombinant expxession ho t or alteration in the glycosylati~ n ites. Forrns of the glycoprotein with ;: Epecified glycosylation patterns will be so ~oted .
As :used herein, the a subunit "minigl~ne" refers to ~ithe géne constru~tion disclosed ~ in PCT applicatiorl WO
o ~ 90/0980:0 in the description of the coxl~;truction of ,.~, 12/CGa or p~l2/cY. ~This "minigene" i5 characterized by rete~tion only ~of 'one iiltron sequence such as that between exon III and~ exon IV, all other introns ha~ring been~;deleted:.~In the particular construction described, t~e~N-terminal:co~ing sequenc~s which are derived from ~: exon II and a~portion of exon III are ~pplied from cDNA
: a~d arè ligated directly through an ~baI re triction site : into the coding se~uence ~f exon III 80 that the i~tron~
between exons I and II and between exons II and III are absent. Howe~er, the intron between:exons III and IV as well as the signals 3~ of the coding ~equence are :: retained. The resulting minigene can con~eniently be ~:: inserted as a BamHI/BglII segment. Other means for ~ construction of a comparable minigene are, of cour~e, :: :
wo 93/2lgq7 2 i 1 8 3 2 0 P~T/U~93/OqOSl possible and the definition is not restricted to the parti~ular construction wherein the coding se ~ ences are ligated through an XbaI site. However, this is a con~enient means for the construction of the gene, and S there i~ no particular advantage to other approaches, such as synthetic or partially synthetic preparation of the ge~e. The definition includes those coding se~uences for the ~ subunit which retain one intron such as t~at between exons III and IV but no other introns.
~: 10 A~"tran~formed" recombinant host cell, i.e., a cell ~transformed~ with the recombinant expression ~ystem~ of:the inve~tion, refers to a host cell which has bee~ alt~red to contain this expre~ ion sy~t~m by any conveni~nt manner of introducing it, including ~:~:; 15 tra~sfection, viral infection, and 80 forth.
: "Tra~sfonmed" refer~ to cell~ co~taining this expression system wh~ther the ~ystem is integrated into the chro~osome or is~extrachromosomal. The "transform d~
cells:may either be stab~e with re~pect to inc~usion of 20:~ ~the expression system or not. In~short, recombînant ~st cells~"transformed" w~ h the expre i~n ~y~tem-of the : invention ref rs:t~ cells which include thi~ expres~ion ystem as a; re~ult~ of their manipulation to include it, when they na~i~el~:do not, regardle~s of the ma~ner of 25 ~: ef~ect~ing ~his inco~poration.
Expression ~y~tem" re~er to a DNA sequence which i~cludes a coding sequence to be expre~ed and : tho~e accompanying control D~A sequences nece~ary ko effect the expre~sion of the coding ~e~ue~ce. Typically, 30~ these control inc1ude a promoter, termination regulating equ~nce~, and, in ~ome cases,~an ~perator or other mechani m to regulate expression. Th co~trol sequences are tho~e which are designed to be functional in a particular target recombinant host cell and ~herefore the WO93/21947 . . PCT/US93/04nSI
host cell must be chosen ~o ~s to be compatible with the control sequences in the constructed expression system.
As used herein "cells~ 'cell cultures", and : "cell lin~" are used interchangeably without particular attention to nuances of meaning. Where the di~tinction etween them is important, it will ~e clear from the context. Where any can be meant, all are intended to be include~.
Certain cells are known to contain dense-core ~secretory gr~ules and to secrete proteins through a regulated pathway, which can be stimulated by certain ~ubstances, for example, forskolin. These cells or cell Iine , deri~èd from appropriate animal ti~ues, are the ho~t celIs of the invention. Included among cuch cells :l5~ are cells of~the secretory component~ of ~he hormo~e y tem~uch;as~:the:pituitary, ~ i let cells, and cells of the~adre~al co~tex:.:~ Particularly preferred in the method of; the invention are pituitary-derived cell~.
o~ tent~with the foregoing paragraph, "cell~
2:0~ ;deri~ed rom:pituitary" refers:the cellR or cell lines ;which~are cultured~fr~m pituitary tissue:derived from ani~al~ pe:cies,; in particular;mammalian specie~, and more paxticulàrly,:~hu~ar or:murinç;pituitaries. Illustrated herein i~the G~3 murine~cell~line described by Tasjian, 2~5~ :J.,~ (197:9):~ 5~7.: However, other lines derived:from~the:pituitary~are~al~o ~nown and obtainable rom p ~ lic depo~itories. In addition, cells deri~ed directly from~pituitaries:may~be~:~u~ed. ~ ~
Cell~s containing ~ecret:ory::granules provide an ;30~ environment:more like that :o~ ~pituit~ry cells as opposed to CHO cell~. This re~ultæ in glyco~ylation patterns : : more like the nati~e orms~:and enhances ~ecretion efficiency of proteins~to be secreted.~ ~Production of the : gonadotropin~ in such cells,~containing dense-core ::: :
~-.. W0~3/~1~47 ~ 3~ o P~T/U~93/040~1 secretory granules capab~e o secreting proteins through a regulated pathway thus results in the production of secreted forms of these materials with glycosylation patterns similar to those found in the native hormones or subunits. In particular, these forms of the hormones or subunits ~u~h a5 LH which retain labeled sulfur ~upplied in the form of sulfate do so when produced in these cells ~: indicating that ~ulfated glycosylation units are presen~
in the~e forms. ~abeled SO~ 2 i~ not incorporated into : : 10~ ~H produced in CHO cel}s or murine Cl27 cells.
~: While FSH is normally sialylated, the ~ subunit of ~H is normally sulfated. Sialylation of N-linked : : o1igosaccharide~ has long been recognized to pxotect :circulating gonadotropin from clearance by hepatic 15 ~ asialoglycoprotein receptor In contra~t, the pre~ence , ~ ~
` ~:: : ~ o~ulfate on ~H lead to a rapid;clearance of the h~ ormone; ul~ated b~vine LH h~s a 4-5 fold faster ~et~bolic clearance compared to the corre~ponding ialy1at~d~re~ombinant bovine L~I. Sulfated LH is removed 2;0 ~ om~ci~culation upon binding to a ~pecific receptor p~
;the~surface:o~liver epithelial cells which re~ognizes a specific ~ulfa~ed trisacchari~e in ~he~N-linked glycosylation.~;~ De~ulfated ~H~behaves simi~arly to ;:sul~ated~hX~in~recept~r binding:and ignal transd~ction ; 25~ in~:MA-~lO cel~ls,~but~hi~h in~ vivv do9es are requixed to ti~ulate o~ulation,~pre~umably be au e ~he circulatory half life is altered by sulfation. It is believed that clinical u~e of ~R:in human~or animal reproduction wilI
requlre the hormone~in~sulfated form.
, 3:0~ ExPression Vectors ~ ~
:~ ~To co~struct ~uitable:expression vectors for , use in the ~ecretory cells of the invention, a convPnie~t co~struct,~ iIlustrated for the ~ subunit minigene i5 ~::
:
WO93/~1~47 21 1 832 0 P~/U~93/~051 :~
reproduced from the above-referenced PCT application as Figure 1. As shown in this figure, and as more fully explained in the above-referenced app~ica~ion, the se~uence encoding the ~ sllbunit is prepared as a minigene wherein the portions of the peptide encoded by exons II-III are fused but separated from exon IV. Thi~ construct : is ligated into the host vector pM2 under the control of the long terminal repeat ~hown in Figure 1 to obtain : pM2/CG~ or to obtain pM~a which contains an additional ~0 insertion site for another coding D~A. As further shown in Figure l, the BamHI site contained in the ho~t vector pM2 can be u ed to accommodate the ~ subunits of the human r productive proteins for expre~sion of the~e subunit per~ se, a well a~ providing a separate ; 15 ~expr~ssio~ vector for concomitant production of the a and subunit by rells tran~fo ~ ed by both vectors.
Thus, the:~ sub~nits per se may be produced by ligating the coding DNA ~into pM2 a~ ~hown, or the heterodimer may be produced by cotra~sfo ~ ation of pM2 :~aontainîng ~A;encoding ~ ~ubu~it with pM2/CG~
Production of ~he;heterodimer i~ preferred. The heterodimer may:also~be~produced from the;single vector wherein the ~ in ert i liyated into the Bam~I site of pM2/a~créated~by ligation of EcoRI-digested pM2 with 25~ ~co~ dige~ted modified pM /CG~ -:~: The ~oregoing construc~ions are, of cour e, ' ~ merely illu~trative of expression vectors or ~ystems~
: : which an be constructed for the prod~ctio~ of ~ subunits or the correspondi~g:heterodimeric hormones. Alternate ;: : 30 :control ~equences incIuding, for exa~ple, different : ~ promoters, can be ligated to the coding seque~ce of human ubunit~ or the a minigene to effect expression. A
~:~ : ~ariety of control sequences is known in the art, and methods to ligate the ~ subunits or ~ minigene coding ~ :
W093/21947 2118 3 ~ ~ PCTlUS93/~51 sequence (or other ~-encoding construct) are of course also available~ Suitable mammalian promoters include the early and late promoter~ from SV40, or other viral : promoters such as those derived from polyoma, adenovirus : 5 2, bo~ine papilloma virus or avian sarcoma viruses.
: ; : Suitable viral and mammalian enhancers can also be used.
~ As ~et forth in the Background section above, ;~ the recovery of the genes for the various ~uman :
reproducti~e hormones, including their ~ subunits, has : 10 already been described. The genes can be recovered from native ~ource~ as described in the art, or the genes can be entirely or partially synthesized u~ing ætandard solid phase oligon~cleotide ~yntheæis techniques as described, :;for example, ~y Nambiar, K.P. et al. Science (~984) 5;~2 1299 or by Jaye, E. et al. J Biol Chem (19843 259::6311. The ~echniques are now commercially ;ava~ilable. It~i~ evident, of cour~e, that not only th~
specific native nucleotide ~equence~ can be employed, but al~o nu:~eotide equences:employing codons which are o~ degenerate~with thes~e~,~ as~well:~a~s~allelic~forms. .~-~: AB f~rther~de~cribed~in the aboYe-referenced PCT~applicatio~n,:muteins of the:variou~ hormone~ can be o~tained~:which:have~agoni~t or antagoni~t activity.
These~mutein-encoding~genes~may~be inserted into the 25 :~ rele~ant ho t:vectors in a manner~:similar to that de~cribed for~the nati~e for~s. Of parti~ular intereæt : are mutein wherei~ the:~ subunits of LH, TSH or FSH are : extended b~ the carboxy:terminal peptide native to :: : chorionic gonadotropin.: One ~uch ~on~truct is 30~ lu~trated in Figure 2 for human~F5H. In addition, more than one of t~e ~TP~uni~s may be used to extend the : chains.
: , WO93t21947 ~ 3 ~ O PCT/U~g3/~051 In general, the selected expression v~ctor or vector~ is transfected or otherwise transformed into the host cells of the in~ention using procedures similar to ~:S those u~ed generally ~or mammalian cells. The techniques ~: ~for tran~formation are ~ubstantially those applied with re~pect to Chine~e hamster o~ary cells, and ~y~tems for marker~ for selection of succe~ful transformants are : ~- a1so ub~tantially~identical. Cell culture conditions for the ~ransfonmed cells ma~ be---modified as is generally under tood to accommodate ~he particular need~ of the selected ho~t cell line which will contain reyulated secretory g~anuleæ; however, culture conditions are ` 8ub8ta~tially similar to tho e u ed for ~HO cells a 15~ well. ~
In an illustrative approach to transfection and cultu~e, the:~subunit-encoding yene i9 inserted into pM2 an~then transfected alone or along with~pM3/CG~ into the ce~ ;appropriate for the method of the:invention which `O~ con~ain regulated ecretory granu1es or into Chinese ~-~iham~ter~o~ary:cells as~a contr~l,~a~ described by Matzuk, M.M.~ et~al., ~b=~ g~bS~ L (;1987) 8~:6354-6358;
Matzuk~ M.M.~ e~al., ~ 5~LL~al:(19883 106:lO49-1059.
Succe~ful transformant :are ~elected in 0.25 ug/ml G4l8, 25:~ and~e~pre sion~may~be~detected ~y~immunoprec:ipitation of : metabolically labele~ cells to elect:monomer- and dimer-secreting cell lineB.
- Both~stably tran~fectQd~hosts ~ontaining resulated secretory granule and:~tably tran~fected ~HO
: 30~ cell~;lines are maintained in "medium-~l" (Ham'~ FlO medium æupplemente~ with 1~5% horse serum`and 2~5~ fetal calf :
erum, penici1lln (100 U/ml), streptomycin (lO0 ~g/ml), :glutamine (2 mM) and 0.125 mg/ml G~l~ in a humidified 5 ,~
:C2 incubator.
~ .
.. WO93/21947 2 1 18~ ~ P~T~US93/0~51 Labelinq Both cells of the invention and C~0 cells are plated at 300,000-350,000 cells per well into 12-well di~hes in 1 ml of F-10 medium containing 15~ horse serum ~ ~ S and 2.5~ fetal calf serum and pr~plated 3-4 days. For : : : continuou~ labeling, cells are washed twice with ; cysteine-frèe "medium-2" (medium~1 supplemented with 5~
dialyze~ calf ~erum) and labeled for 6 hours in 1 ml of cysteine-free~medium 2 containing 20 uCijml la~eled 0 ~ cyseeine. ~For pulse cha~e experimentæ, the cells are washed twice and preincubated for 1.5 hours in cy~teine-~ree medi~m-2,~fo~1Owed by a 20 minute labeling in cy teine-fre@~medium-2 contai~ing 100 uCi/ml labeled :cy~teine,~ then washed twice with medium 2:containing 1 mM
5~ unlabeled~cyst:eine and incubated in the unlabeled medium.
a~elin~ ~periment~ using incorporation o 35S:-labeled sulfate~are~aonducted similarly.
In~more detail the medium`and cell ly~ate are prepare~, immunopre~ipitated, and:treated~as described by 2~ ~rle~6,~ c.~ et al. ~ J e1l ~ioI~ 87): 104:1173~
and~ he~Matzuk~P~a~ paper cited~above. Anti~era agalns~ G~ ~H~ :FSH-~ and T9H~ and the ~ ~u~unit are preparèd~by~tandard:methodsi:~antisera~generated against CG-~cro~s~-~reaetæ ~ully~with ~ a~d~can be u~ed to 25;~ ;detect LH-~as well.~ ~For ~haracterization of the : immun~precipi~ate ~n SDS ~els,~ SDS~polyacrylamide gel are soaked for~10 minutes in l M~sodium ~licylate, d~ied~ and;autoradiographed~with preflash film,: ~nd scanned, i~: desired, with:an LKB~Ultra~can X~ lasex:
3~0:: ~den~itometex.~
: As~a~cor.trol, the results~:of production of : human FSH;in:CHQ cells is asseæsed by l~elin~ with ~: ' :
::
WO93/21947 2 i 1 8 3 2 0 PCT/US93/~1 cy teine. The expres~ion systems described above for human FSH-~ inserted into pM2 for expression of F5H-~alone or into pM2/~ for expre~sion in tandem with the ~
subunit were transfected into CHO cells and stable clones shown to e~pregs the ~ subunit or dimer were continuously labeled with 35S-cy~teine for 6 hr. Yields of secreted FSH of approximately 1 mg/106 cells/24 hr cultured in 1 L
of medium were obtained.
: The proteins secreted into the media and from cell ly~ates were immunoprecipitated with appropriate anti~era and resolved on SDS-PAGE. The results obtained : com~are the behavior o~ similar transformants expressin~
the gene for human C~
Gel~ from 6 hr labeling ~how that in the ~abQence of ~ subunit FSH-~ i9 retained in the ly~ate, while when the:~ ~BUbUnit i~ pre~ent, the dimer is formed ;and efficie~tly ~ecreted into the medium.
When the cells are pulse~labeled wit~
35S-~cysteine~for~20~min and:chased with unla~eled 2~ ¢y~teine for up~to 12 hr,~result~ for the ~ subunit of~CG
show~the~half-life in lysat~es and appearance of-CG-~ in t~e~medium are~identical;at about 2~hr and almo&t all the secre~ed~ ubunit can~:be recovered:. A lower molecular weigh~ present ~in the:~medium a~parently due to the 25~:~ dif~ereDces; n the extent:of:glycosylation at the 2 APn-:: linked~glyco ylation:sites on CG-~ and is unigue to this ubunit. FSH-~ alone is secreted much les~ efficiently and, a~ does~G-~, di~appear~ from the cell lysates af~er a~out 5 hr; 1 ss than 20~:is recovered in the medium : 30 after 1~ hr.
Simi:larly~:to the ~ subunit~ of:LH and TS~, FSH-alsne is ine ficiently secreted and slowly degraded intracellularly. : Howe~er, the pre~ence of the ~ subunit tabilizes and enhances the secretion of the ~ subunit W093/21947 2 1 1 ~ 3 2 O PCT~US93J04~51 -l5-for FSH. The half-life for diRappearance from the lysates was about 90 min, and 90% was recovered in the medium after l2 hr. This behavior is similar to that shown for TSH, but different from both CG and LH.
I~ i~ apparent from the foregoing results that dif~iculties with ~ecretion are experienced or expression in CHO cells of the genes encoding the ~
: subunits alone; in ome instan~es, this secretion abili~y is mitigated by the presence o~ a vector encQding the ~
~ ~ubunit. However, in general, it appears that CHO cells are not efficient secretors~of the desired ~ subunits or correspondi~g human reproducti~e ho ~ one~
AnQther~difficulty which is apparent from experiments using sulfate label as a source of 35S is ;;l5~ that~for L~ subunit or thP LH heterod.imer, which is k~own to contain ulfated glycosylation in it6 native tate, in~orporation~of label does not take place in CHO
G~ h~s, :~a~ lea t~for LH, and~for:the ~ portion of th~ :heterodimer., which~also contain~:sulfated 2~0~ glyco~ylation~,~HO~;cel~lc sre unable to reproduce the _-~
b~ native::patte~n::for glyco~yla~io~.: Thus, it has been show~ that u~ing,~;~in~place;of ~ysteine, labeled sulfate a~:;a ~ource:o ::ra~ioacti~ity, label does not appear in the~di~er~or~ the ~ hunit of~ L~ produced in CHO cells~
:: 25 ~ ~
: The~:e~pressio~ vectors described aboveincluding :the~ ubunit~ for LH~ CG-~ or FSH-~ either with or without the~a subunit,:and~with the ~:subunit encoded on the same~or different ve~tor wexe tran formed 30~ ~into the:rat~ pituitary cell~ line GH3. After æteady s~tate : :laheling ~y either sulfate (for ~H~ or cys~eine-oriyinated ~for all~:forms) S35,~mature forms of LH, FS~
and hC~ which contained processed oligosaccharide~ were~
~: : :
W~93/21947 2 1 1 8 3 2 0 PCT/U~93/~4051 ~tored in the cells and their release was stimulated by for~kolin. The majority of the ~ ~ubunits of these hormones appeared to be Endo H sensitive and thus re~ide in the ER, but the Endo H resi~tant fraction, which i6 the mature form, was targeted to the regulatory ~ecretory :: pathway with an efficiency that was comparable between : the dimer and the ~ ~ubunit.
Figure 3 shows the result~ obtained from G~3 cells which have been tran~formed with pM2/~ into which ~: 10 the coding ~equence for hH-~ ~ubunit has been inserted into the alternate BamHI site. Lanes 1 and 2 are controls f~om ~H3 ~ell~ which are trans~ormed only ~y pM~/CG~; a i~ evident from the results, the ~ ~ubunit is found mo~tly in the cells themselve~ (C) rather than in : lS ~the ~up#rnate (S? ~n the other hand, lane~ 3 ànd 4 : repre~nt the re ults obtained b~ immunoprecipitating the :~ .
he~rodi~er labeled as de ri~ed above u~i~g cysteine or ul ate~and a~aying the cell medium. A~ ~een from these re~ult~ a large amount of the heterodimer i~ ~ecreted 20~ into~the medium and both the ~ and ~ ~ubunit are able take up }abel rom~5 sulfate.:
n an;additional e ~ eriment, cell~ expres~ing ; :LH were la~eled with~t35S]04 for 16::~rs~to allow accu~ulatio~ of the labeled hormone~ in ~he ~torage :~gran~les. To:~de~crea~e the background of ~onstitutive ~ecretion (i.e., the material not ~ecreted through den~e core granules of the reyulated pathway) following the ab21i~g, the cells were cha ed twice at 2 hr i~tervals :
befo~e timulation (desi~nated I and II in ~igure 4). ~t : 30 the beginning o the:third cha~e (III), 20 ~M of the : ~ secretagogue forskolin (an adenylatecyclase activator) : was added to a duplicate set of wells; a parallel ~et was ~ incubated without ecretagogue. The media (M) and the . W~93/21947 2118 3 2 0 PCT/US93/04~1 cell extracts (L) ~ollowing the third cha~e were precipitated ~y an~ serum and analyzed on SDS-PAGE.
In Figure 4, panel A, lane 1 represen~s the medium at cha~e I. This lane shows that LH~ and ~
subunit~ wexe labeled~by [35S]04. This initial pool is presumably a rapidly secreted, not stored pool. After 4 : ~ ~, hours (II~ and 6 hours (III) of cha~e, the constitutive pool is depleted i~ce no further material is prese~t in the media ~1~a~es 2 and 3 of panel A). Howe~er, there is stil1 a pool of ~H i~. the cell as ~hown by panel A, lane 5 which repre~ents tn~ lysate of cells not treated with : forskoli~. Thi~pool is r~.-lea~ed by the secretagogue :; into the ~edia as shown by 2anel A, lane 4. Lane 6 shows that the intr~cellular pool is depleted following ~orskoli~ treatment~
To demo~strate that radioactive ~ulfate wa~
corporated~i~to N~ ked oligosaccharide~, immunoprecipitated~35S]04 labeled LH was treated with endoglycosida~e~F which is known t:o remove c~mplex N-20~ inked 0ligo~accharides. ~The results of thiæ treatmç~are shown::in~Figure 4, panel B, lane~ I and 2.~ Upon digestion, with~Endo F, the~radioactivity disappeared fo~m~both~a~and~sl~hunits. ~anes 3 and 4 are controls to~how that~t~i~ wa~ not due:to protease activity during :25 ~ incubation.:~ The~e~lanes show the result of Endo F
treatment of LH ~ecreted:into ~he medium after labelIng : ~ with 35S-cy teine. The 35S-cy teine labeled LH subunits collapRe to~a~sin~le band orregponding to the deglycosylated;protein following Endo F dige~tionO
:~ ~ ~ The~e data show that~ LH iæ ~tored in the : ~ ~ GH3 cells~and it ~:release ls:s~imul~ted by 3ecretagogue uch ~torage and:release is not 9een in CH0 cells :~ whethex ~a~eled with S04 or cysteine), (2~ The N-linked :: :
W093~2~947 2 1 i 8 3 2 0 P~T/US93/04~1 .
oligosaccharides of LH are sulfated when produced in GH3 cells.
As~embly of the ~ 3ubunits of these hormones and secretion of the dimers is increased many-fold over that en in CHO cells. The oligosaccharide in the LH
dimer is al~o ~ulfated; howe~er, FSH is normally sialylated in~ ~o,' and thus does not take up label from S04~ ~ :
It has b~en demon~trated that complete 10 ~deglycosylation of human chorionic gonadotropin results in a hormone which retain~ its ability to ~ind to receptor, but :is no longer capable o~ eff~cting the ordinary biological:~re~pon e of the cell on which the receptor i borne. Simila~ effects of ~omplete.
15~ eglyco~ylat;io~are~o~tained with the additional reproductive;~hor~one. LH and FSH.~ Accordingly, alt~ration o the glycosylation::pattern in the ~ subunit may~;:ra ult in:alternative~propertie~. The glycosyl~tion of;the~:hormones an~:~subunits produced b~he method of 2~ he~i~ve~ however;~ more clo~ely:re~emble~ hat o~-the~native~iorm~
The:~honmone~and~oth~r~pharmace~tical of the~
presen~inYe~ion~are formulated ~or~administration u ing 2~ ~method ~generally u~der~tood in~the a~t~ Ty~ical : ; formiulation a~d ~odes of:admi~i tration are descrihed in Mack~Pu~lishing Co., Easton, PA,~ latest~edition.~The~e~formulation~ are typically ~or~ystemic:administrati~:, such as by 30~ :i~jection, but oral~f;ormulations~:or topical ~ormulations may al~o be~employed.
The choice of formul~a~ mode of admini~tration,:and do~age lèYel are ~dependent on the .
:
WO93/21947 2 ~ 1 8 3 2 0 PC~/US93/~4051 particular hormone or protein and can be optimized for the appropriate indication u ing generally recognized techniques.
:
~: :
:
::
~: :
,.
~ . : : . :
:: ~: :
:
Claims (8)
1. An improved method for recombinant production of a human glycoprotein selected from the group consisting of hCG, FSH, LH and TSH, which method comprises culturing animal cells that contain regulated secretory granules, which cells have been modified to contain an expression system capable of expressing a DNA encoding said glycoprotein or the .beta. subunit thereof under conditions wherein said encoding DNA is expressed, and recovering the glycoprotein or .beta. subunit thereof from the culture medium.
2. The method of claim 1 wherein said cells are pituitary cells.
3. The method of claim 2 wherein said pituitary cells are GH3 cells.
4. The method of claim 1 wherein the glycoprotein is LH or FSH.
5. A cell culture capable of secreting a human glycoprotein selected from the group consisting of hCG, FSH, LH and TSH, which cell culture comprises animal cells that contain regulated secretory granules which cells have been modified to contain an expression system capable of expressing a DNA encoding said glycoprotein or the .beta.
subunit thereof.
subunit thereof.
6. The culture of claim 5 wherein said cells are pituitary cells.
7. The culture of claim 6 wherein said pituitary cells are GH3 cells.
8. The culture of claim 5 wherein the glycoprotein is LH or FSH.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US07/876,794 US5985611A (en) | 1992-04-30 | 1992-04-30 | Recombinant production of gonadotropins in secretory cells |
US07/876,794 | 1992-04-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2118320A1 true CA2118320A1 (en) | 1993-11-11 |
Family
ID=25368600
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002118320A Abandoned CA2118320A1 (en) | 1992-04-30 | 1993-04-30 | Production of reproductive hormones |
Country Status (12)
Country | Link |
---|---|
US (1) | US5985611A (en) |
EP (1) | EP0644774A4 (en) |
JP (1) | JPH07506968A (en) |
AU (1) | AU681849B2 (en) |
CA (1) | CA2118320A1 (en) |
CZ (1) | CZ284885B6 (en) |
FI (1) | FI945037A (en) |
HU (1) | HUT69984A (en) |
NZ (1) | NZ252484A (en) |
SK (1) | SK130894A3 (en) |
WO (1) | WO1993021947A1 (en) |
ZA (1) | ZA933070B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014159813A1 (en) | 2013-03-13 | 2014-10-02 | Moderna Therapeutics, Inc. | Long-lived polynucleotide molecules |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2710845B1 (en) * | 1993-10-08 | 1996-03-29 | Lafon Labor | Composition intended for the immunotherapy of a cancer secreting hCG or fragments of hCG. |
US7081446B2 (en) * | 2002-01-31 | 2006-07-25 | The Trustees Of Columbia University In The City Of New York | Long-acting follicle stimulating hormone analogues and uses thereof |
US7173113B2 (en) * | 2002-01-31 | 2007-02-06 | The Trustees Of Columbia University In The City Of New York | Long-acting hormone and growth factor compositions and uses thereof |
DE602004030546D1 (en) | 2003-03-04 | 2011-01-27 | Aspenbio Pharma Inc | LH for use in maintaining one or more pregnancies by inducing the formation of the secondary corpus luteum. |
JP4412989B2 (en) * | 2003-12-15 | 2010-02-10 | 株式会社日立製作所 | Data processing system having a plurality of storage systems |
BR112021022860A2 (en) | 2019-05-16 | 2022-01-18 | Ceva Sante Animale | Compositions and methods to enhance reproductive performance in non-human mammals using recombinant luteinizing hormone |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US4828987A (en) * | 1984-05-14 | 1989-05-09 | Merck & Co., Inc. | Avian retrovirus-bovine growth hormone DNA |
CA2053864C (en) * | 1989-02-21 | 2001-11-20 | Irving Boime | Modified forms of reproductive hormones |
-
1992
- 1992-04-30 US US07/876,794 patent/US5985611A/en not_active Expired - Fee Related
-
1993
- 1993-04-30 JP JP5519520A patent/JPH07506968A/en active Pending
- 1993-04-30 EP EP93910915A patent/EP0644774A4/en not_active Ceased
- 1993-04-30 HU HU9403094A patent/HUT69984A/en unknown
- 1993-04-30 ZA ZA933070A patent/ZA933070B/en unknown
- 1993-04-30 NZ NZ252484A patent/NZ252484A/en unknown
- 1993-04-30 AU AU42241/93A patent/AU681849B2/en not_active Ceased
- 1993-04-30 SK SK1308-94A patent/SK130894A3/en unknown
- 1993-04-30 WO PCT/US1993/004051 patent/WO1993021947A1/en not_active Application Discontinuation
- 1993-04-30 CZ CZ942662A patent/CZ284885B6/en unknown
- 1993-04-30 CA CA002118320A patent/CA2118320A1/en not_active Abandoned
-
1994
- 1994-10-26 FI FI945037A patent/FI945037A/en not_active Application Discontinuation
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014159813A1 (en) | 2013-03-13 | 2014-10-02 | Moderna Therapeutics, Inc. | Long-lived polynucleotide molecules |
Also Published As
Publication number | Publication date |
---|---|
FI945037A (en) | 1994-12-21 |
HU9403094D0 (en) | 1994-12-28 |
NZ252484A (en) | 1996-06-25 |
ZA933070B (en) | 1994-05-03 |
AU681849B2 (en) | 1997-09-11 |
AU4224193A (en) | 1993-11-29 |
CZ284885B6 (en) | 1999-03-17 |
US5985611A (en) | 1999-11-16 |
FI945037A0 (en) | 1994-10-26 |
HUT69984A (en) | 1995-09-28 |
EP0644774A4 (en) | 1997-03-12 |
JPH07506968A (en) | 1995-08-03 |
WO1993021947A1 (en) | 1993-11-11 |
EP0644774A1 (en) | 1995-03-29 |
CZ266294A3 (en) | 1998-11-11 |
SK130894A3 (en) | 1995-07-11 |
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